Pyrrolo[3,4-c]pyrrole-1,4-dione, 3,6-bis(4-chlorophenyl)-2,5-dihydro-

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 1986-02-25 to 1986-03-25

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP guideline study according to OECD guideline 301 B; the volume of the test solution was reduced from 3.0 L to 1.5 L, no blank control was conducted

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

1981

Deviations:

yes

Remarks:

, the volume of the test solution was reduced from 3.0 L to 1.5 L, no blank control was conducted

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Sewage treatment plant in CH-4153 Reinach on 1985-03-12

Duration of test (contact time):

28 d

Initial conc.:

10 mg/L

Based on:

test mat.

Initial conc.:

20 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Test temperature: 22 ± 2 °C
- Aeration of dilution water: Approx. 50 mL/min free of carbon dioxide

TEST SYSTEM
- Culturing apparatus: 2 L flasks equipped with gas inlet and magnetic stirrer.
- Number of culture flasks/concentration: 1
- Details of trap for CO2 and volatile organics if used: Absorbers filled with 0.025 N Barium hydroxide
- Measuring: Titration of CO2

SAMPLING
- Sampling frequency: Days 6 / 9 / 13 / 16 / 20 / 23 / 27 and 28

CONTROL AND BLANK SYSTEM
- Inoculum blank: None
- Reference substance (Aniline)

Reference substance:

aniline

Remarks:

20 mg/L

Parameter:

% degradation (CO2 evolution)

Value:

10

Sampling time:

28 d

Remarks on result:

other: Cumulative result for 10 mg substance/L

Parameter:

% degradation (CO2 evolution)

Value:

4

Sampling time:

28 d

Remarks on result:

other: Cumulative result for 20 mg substance/L

Details on results:

The biodegradation was calculated on the basis of the theoretical carbon content of the test substance and the cumulative quantities of carbon
dioxide determined on the days of measurements. For the calculations the formula given in the guideline was used.

Results with reference substance:

The biodegradation was calculated as: 105 % in 28 days (20 mg Aniline/L)

Table 1. Carbon dioxide values and biodegradation cumulative in % CO2/TCO2

Days	Test substance				Aniline
	10 mg/L/mg CO2	% degrad.	20 mg/L/mg CO2	% degrad.	20 mg/L/mg CO2	% degrad.
6	0.0	0	0.0	0	6.6	8
9	0.93	3	1.3	2	48.7	65
13	0.0	3	0.0	2	9.8	77
16	0.4	4	0.4	3	6.1	84
20	1.3	8	0.6	4	7.0	94
23	0.0	8	0.0	4	3.1	96
27	0.7	10	0.6	4	2.9	99
28	0.0	10	0.0	4	5.2	105

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test substance is not readily biodegradable (by OECD criteria).

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 1986-02-25 to 1986-03-25

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP guideline study according to OECD guideline 301 B; the volume of the test solution was reduced from 3.0 L to 1.5 L, no blank control was conducted

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across is performed between two forms of the same substance. The identities of the two forms are describe below.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source form is 3,6-bis(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrol-1,4-dione (EC-no. 401-540-3), referred to here as PR254. PR254 is an organic mono-constituent substance with a typical purity of > 99.5% (w/w). It does not contain any impurity relevant for classification or labelling of the substance. The target form is the nanoform of the source substance, referred to here as PR254 nanoform. As the source form, it has a typical purity of > 99.5% (w/w) and it does not contain any impurity relevant for classification or labelling of the substance. The PR254 nanoform is spheroidal with a pure polyhedral shape and is not surface-treated.

3. ANALOGUE APPROACH JUSTIFICATION
The two analogue forms have the same structure. Under ambient atmosphere, the specific surface energy of particles increases with decreasing particle size. Therefore, particle aggregate to reach an energy minimum. The driving forces are hydrogen bonds and van der Waals forces (π-π interaction). Substantial energy is required to disperse the PR254 nanoform aggregates to particles that fall under the nanoform definition.
PR254 was been tested extensively addressing information requirements of Annexes VII to IX without identifying any biological target. PR254 nanoform could potentially have biological targets due to the different particle size distribution, which would require processes capable of dispersing the aggregates, e.g. in aqueous milieu. However, both forms have been tested according to OECD Test Guideline 318, demonstrating that PR254 nanoform cannot be dispersed under the condition of the study, i.e. immediately after sonification re-forms aggregates. Also, PR254 aggregates to a large extent, but can be more easily dispersed than the nanoform. The experiments demonstrated that exposure in aqueous milieu will be primarily to aggregates, regardless of the PR254 form.
Therefore, it is concluded that both forms will behave identically in studies, in which they are applied under atmospheric conditions and/or in aqueous milieus, so that for the PR254 nano-form no specific biological targets need to be considered.
As both forms are hardly dispersible and have a very low water solubility, the aggregates are stable in the environment, i.e. the forms do not hydrolyse and do not disperse. As the stability of the aggregates of the PR254 nanoform is even higher than that of the PR254 bulk form, read-across from the source to the target form is conservative and scientifically justified. Biodegradation, bioaccumulation, and environmental transport and distribution can be assumed to be identical.

4. DATA MATRIX
The data matrix is included as Annex 1 in the assessment report ‘PR254 bulk nano analogue approach 210111’ attached here below under ‘Attached justification’.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

1981

Deviations:

yes

Remarks:

, the volume of the test solution was reduced from 3.0 L to 1.5 L, no blank control was conducted

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Sewage treatment plant in CH-4153 Reinach on 1985-03-12

Duration of test (contact time):

28 d

Initial conc.:

10 mg/L

Based on:

test mat.

Initial conc.:

20 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Test temperature: 22 ± 2 °C
- Aeration of dilution water: Approx. 50 mL/min free of carbon dioxide

TEST SYSTEM
- Culturing apparatus: 2 L flasks equipped with gas inlet and magnetic stirrer.
- Number of culture flasks/concentration: 1
- Details of trap for CO2 and volatile organics if used: Absorbers filled with 0.025 N Barium hydroxide
- Measuring: Titration of CO2

SAMPLING
- Sampling frequency: Days 6 / 9 / 13 / 16 / 20 / 23 / 27 and 28

CONTROL AND BLANK SYSTEM
- Inoculum blank: None
- Reference substance (Aniline)

Reference substance:

aniline

Remarks:

20 mg/L

Parameter:

% degradation (CO2 evolution)

Value:

10

Sampling time:

28 d

Remarks on result:

other: Cumulative result for 10 mg substance/L

Parameter:

% degradation (CO2 evolution)

Value:

4

Sampling time:

28 d

Remarks on result:

other: Cumulative result for 20 mg substance/L

Details on results:

The biodegradation was calculated on the basis of the theoretical carbon content of the test substance and the cumulative quantities of carbon
dioxide determined on the days of measurements. For the calculations the formula given in the guideline was used.

Results with reference substance:

The biodegradation was calculated as: 105 % in 28 days (20 mg Aniline/L)

Table 1. Carbon dioxide values and biodegradation cumulative in % CO2/TCO2

Days	Test substance				Aniline
	10 mg/L/mg CO2	% degrad.	20 mg/L/mg CO2	% degrad.	20 mg/L/mg CO2	% degrad.
6	0.0	0	0.0	0	6.6	8
9	0.93	3	1.3	2	48.7	65
13	0.0	3	0.0	2	9.8	77
16	0.4	4	0.4	3	6.1	84
20	1.3	8	0.6	4	7.0	94
23	0.0	8	0.0	4	3.1	96
27	0.7	10	0.6	4	2.9	99
28	0.0	10	0.0	4	5.2	105

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test substance is not readily biodegradable (by OECD criteria).

Reference 3

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP guideline study according to 84/449/EEC,C.5; Modified Sturm test; Adaptation of inoculum was conducted according to OECD 302B (Zahn-Wellens); Volume of the test solution was reduced from 3L to 1.5L; Due to poor water solubility of test substance, no stock solution was prepared and an emulsifier was used to achieve a better distribution in the medium.

Qualifier:

according to guideline

Guideline:

other: 84/449/EEC,C.5; Modified Sturm test

Deviations:

yes

Remarks:

, see "Principles of method if other..."

Principles of method if other than guideline:

- Adaptation of inoculum was conducted according to OECD 302B (Zahn-Wellens)
- Volume of the test solution was reduced from 3L to 1.5L
- Due to the poor solubility of the test substance in water, no stock solution was prepared and an emulsifier was used to achieve a better distribution in the medium.

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

- Source of inoculum/activated sludge: Sewage treatment plant of CH-4153 Reinach on 09/03/93
- Preparation and preparation of inoculum for exposure: The preparation was carried out according to the method described in the Zahn-Wellens test (OECD 302B). The concentration in the 2 test vessels was adjusted to 1.0 g/l (dry weight of inoculum). After a preadaptation period of 21 days with the test substance at an initial concentration of 100 ± 5 mg/l the biomass was collected for the inoculum in the CO2-evolution test. The final concentration in the CO2-evolution test was 25 mg/l suspended solids (dry weight).

Duration of test (contact time):

28 d

Initial conc.:

11.5 mg/L

Based on:

test mat.

Initial conc.:

20.1 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Solubilising agent: Polyoxyethylen-Sorbitan-Monooleate (TWEEN 80)
- Test temperature: 22 ± 2 °C
- pH: 7.2
- Aeration of dilution water: ca. 25 mL/min purified from carbon dioxide

TEST SYSTEM
- Culturing apparatus: 2 Liter flasks (dark brown glass) equipped with gas inlet and magnetic stirrer.
- Number of culture flasks/concentration: 1

- Measuring procedure: Determination of the initial CO2 of the 0.05 N sodium hydroxide and the CO2, absorbed in the absorbers filled with 200 ml 0.05 N sodium hydroxide on the days 0, 3, 6, 9, 14,17, 20, 23, 27 and 28,

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Toxicity control: No

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

Remarks on result:

other: Test substance concentration = 11.5 mg/L

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

Remarks on result:

other: Test substance concentration = 20.1 mg/L

Results with reference substance:

106 % biodegradation in 28 days

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test substance is poorly eliminated from water.

Description of key information

The substance was tested in two adapted modified Sturm Tests (OECD 301B), which were reliable with restictions. It was not readily biodegradable in both.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater